In the body, polypeptide drugs are rapidly degraded by proteolytic enzymes and/or neutralized by antibodies. This reduces the half-life and circulation time of the drugs, limiting their therapeutic effectiveness. Attempts to address these limitations have included manipulation of the amino acid sequence of the polypeptide to decrease immunogenicity and proteolytic cleavage, fusion or conjugation of the polypeptide to immunglobulins and serum proteins, incorporation of the polypeptide into drug delivery vehicles for protection and slow release, and conjugation of the polypeptide to natural or synthetic polymers. See, Roberts et al., Advanced Drug Delivery Reviews, 54: 459-476 (2002).
Addition of water soluble polymers or carbohydrates to polypeptide drugs has been shown to prevent their degradation and increase their half-life. For instance, “PEGylation” of polypeptide drugs protects them and improves their pharmacodynamic and pharmacokinetic profiles. See, Harris and Chess, Nat Rev Drug Discov, 2:214-221 (2003). The PEGylation process attaches repeating units of polyethylene glycol (PEG) to a polypeptide drug. PEGylation of molecules can lead to increased resistance of drugs to enzymatic degradation, increased half-life in vivo, reduced dosing frequency, decreased immunogenicity, increased physical and thermal stability, increased solubility, increased liquid stability, and reduced aggregation. Examples of polypeptide drugs conjugated with PEG are PEG-asparaginase, PEG-interferons, PEG-filgrastim and PEG-adenosine deaminase.
PEG is a hydrophilic, uncharged, inert, biocompatible synthetic polymer. In its most common form, PEG is a linear or branched polyether terminated with hydroxyl groups and having the general structure:HO—(CH2-CH2-O)n-CH2-CH2-OH
PEG is synthesized by anionic ring opening polymerization of ethylene oxide starting with a nucleophilic attack of a hydroxide ion on the expoxide ring. An important aspect of PEGylation is the incorporation of various PEG functional groups that are used to attach the PEG to the peptide or protein. Most useful for polypeptide modification is monomethoxy PEG, mPEG, having the general structure:CH3O—(CH2-CH2-O)n-CH2-CH2-OH
Compared with other polymers, PEG has a relatively narrow polydispersity in the range of 1.01 for low molecular weight PEGs (<5 kDa) to 1.1 for high molecular weight PEGs (>50 kDa). Polydispersity is calculated by dividing the weight average molecular weight with the number average molecular weight, and indicates the distribution of individual molecular masses in a batch of polymers (Roberts et al., supra). PEG is soluble in both aqueous and organic solutions, which makes it suitable for end group derivatization and chemical conjugation to biological molecules under mild physiological conditions.
For the coupling reaction between the PEG and the molecule of interest, it is necessary to activate PEG by making a derivative of the PEG having a functional group at one or both termini. The choice of which functional group to activate is depends on the reactive groups on the molecule that will be coupled.HO—(CH2-CH2-O)n-X or MeO—(CH2-CH2-O)n-X
X: functional group for coupling to protein
The most common route for PEG conjugation of proteins has been to activate the PEG with functional groups suitable for reactions with lysine and N-terminal amino acid groups. The monofunctionality of methoxyPEG makes it particularly suitable for protein and peptide modification because it yields reactive PEGs that do not produce cross-linked polypeptides, as long as diol PEG has been removed (Roberts et al., supra).
Branched structures of PEG have also been proven to be useful for PEGylation of a protein or a peptide. For example, a branched PEG attached to a protein has properties of a much larger molecule than a corresponding linear mPEG of the same molecular weight. See, Yamasaki, Agric. Biol. Chem., 52: 2125-2127 (1988). Branched PEGs also have the advantage of adding two PEG chains per attachment site on the protein, therefore reducing the chance of protein inactivation due to attachment. Furthermore, these structures are more effective in protecting proteins from proteolysis, in reducing antigenicity and in reducing immunogenicity. See, Veronese, Bioact. Compat. Polym., 12: 196-207 (1997).
PEG has long been thought to be non-immunogenic. However, it was shown in recent reports that treatment with PEGylated drugs can lead to the development of anti-PEG antibodies in animal disease models and in patients. Those antibodies caused a rapid clearance of the PEGylated proteins from the circulation. Moreover, a considerable amount of anti-PEG antibody in healthy donors was reported in some studies.
To evaluate the incidence of anti-PEG antibodies within a normal, healthy human population, Armstrong et al. [Blood, 102: 556 A (2003)] analyzed 250 plasma samples of normal donors. Anti-PEG antibodies were detected using TentaGel® beads consisting of a PEG-engrafted polystrene matrix. Of the samples analyzed, 25.2% of samples were positive for IgG and/or IgM, of which 18.4% showed IgG binding only, 3.6% IgM only, and 3.2% both. A further study performed in acute lymphocytic leukemia subjects receiving PEG-asparaginase demonstrated the development of anti-PEG antibodies [Armstrong et al., Blood (ASH annual Meeting Abstracts), 108: 1856 A (2006)]. Nine of fifteen subjects who showed undetectable asparaginase activity tested positive by serology and thirteen tested positive when using the flow cytometry, whereas a cohort of thirteen subjects with normal sustained levels of asparaginase activity after treatment did not develop detectable anti-PEG antibodies. In approximately one third of the patients that developed anti-PEG antibodies, rapid clearance of PEG-asparaginase rendered treatment ineffective. No relationship was observed between the presence of anti-PEG antibodies and serum asparaginase activity for patients treated with unmodified asparaginase.
International Publication No. WO 2008/063663 describes assays for detecting anti-PEG antibody in a biological sample.
As alternative to PEGylation, the coupling of polysialic acid (PSA) to therapeutic proteins is used to improve the pharmacokinetic properties. See Fernandes and Gregoriadis, Biochim. Biophys. Acta., 1341: 26-34 (1997) and Gregoriadis et al., Int. J. Pharm. 300:125-130 (2005). U.S. Publication No. 2007/0282096 describes conjugating an amine or hydrazide derivative of PSA to proteins.
Polysialic acid, also referred as colominic acid (CA), is a natural occurring polysaccharide. It is a homopolymer of N-acetylneuraminic acid with α(2→8) ketotsidic linkage and contains vicinal diol groups at its non-reducing end. It is negatively charged and is a natural constituent of the human body.
There remains a need in the art for methods to detect anti-polymer antibodies in individuals who may receive or are receiving water soluble polymer-modified drugs.